miR-100通过靶向IGF2调控非小细胞肺癌A549细胞迁移和侵袭

IGF2-targeted regulation of mi R-100 in migration and invasion of non-small-cell lung cancer A549 cells

目的探讨mi R-100对非小细胞肺癌(NSCLC)细胞迁移和侵袭能力的影响及其作用机制。方法通过Real time PCR和Western blot检测mi R-100和IGF2在人非小细胞肺癌细胞系中的表达;建立了稳定过表达成熟mi R-100的非小细胞肺癌A549细胞系,通过细胞划痕实验和Boyden chamherr检测细胞迁移和侵袭能力的改变;在此基础上,通过报告基因分析和Western blot探讨mi R-100引起非小细胞肺癌细胞生物学功能改变的作用机制。结果 Real time PCR结果表明mi R-100在高转移潜能的非小细胞肺癌细胞A549中呈低表达,而IGF2在A549中呈高表达;在非小细胞肺癌细胞A549中过表达mi R-100后降低了细胞的迁移和侵袭能力,双荧光素酶报告基因分析表明成熟的mi R-100能够作用于IGF2的3’UTR区降低荧光素酶的活性,Western blot进一步证实了mi R-100能够抑制内源性IGF2的表达。结论 Mi R-100在高转移能力的非小细胞肺癌细胞A549中呈低表达,具有类似抑癌基因作用,并调控IGF2表达,降低了非小细胞肺癌细胞A549的迁移和侵袭能力,IGF2是mi R-100的靶基因,有望成为肿瘤生物治疗的靶点。

【Objective】To explore the impact of mi R-100 on underlying invasion and metastasis of non-smallcell lung cancer（NSCLC） and its mechanism.【Methods】The expression of mi R-100 and insulin-like growth factor2（IGF2） in human NSCLC cell line was determined by using real-time polymerase chain reaction（PCR） and Western blot. The A549 cell line stably over-expressing mi R-100 was established by G418 screening. Wound healing and Boyden chamber assays were performed to analyze the migration and invasion of the cells. Based on the changes, the mechanism of mi R-100 involvement in migration and invasion by means of biological function was further analyzed by a dual-luciferase reporter assay and Western blot.【Results】The results of real-time PCR indicated that mi R-100 was down-regulated in NSCLC cell line A549 with high metastatic potential, whereas the over-expression of IGF2 in A549 decreased the migration and invasion. The mutation cell line A549 was successfully established. The wound healing assay and Boyden chamber assay showed that the A549 cell migration and invasion were significantly suppressed. Moreover, the dual-luciferase reporter assay showed that mi R-100 reduced the activity of luciferase in the stable A549 cell line transfected with p MIR-IGF2. This was confirmed by the Western blot analysis, which indicated that mi R-100 suppressed IGF2 expression. 【Conclusions】Mi R-100 is down-regulated in the NSCLC cell line A549 with high metastatic potential. Acting as a tumor suppressor gene, mi R-100 suppresses the migration and invasion by targeting IGF2. IGF2 is a potential therapeutic target for the biological treatment of tumor.